The long-term goal of our research is to understand the molecular mechanisms and cellular processes governing neural tube closure in vertebrate embryos. Our focus is on an essential cell shape change called apical constriction. Apical constriction, a conversion of columnar cells into wedge-shaped cells, facilitates the bending of epithelial sheets and contributes importantly to neural tube closure. We have previously shown that a single protein, Shroom3, is both necessary and sufficient to induce apical constriction in epithelial cells. Shroom3 is also essential for neural tube closure and thus for organogenesis of the brain and spinal cord in frogs, mice and chicks. Recent data demonstrate additional requirements for Shroom3 in epithelial morphogenesis of the eye and the gut. Studies in humans link Shroom3 to renal disease and hypertension. Nonetheless, the mechanisms by which this protein functions remain poorly defined. Here, we propose an integrated approach that will investigate Shroom3 function at the level of fundamental cell biology, tissue morphogenesis, and transcriptional control of gene expression. Recent studies demonstrate that apical plasma membrane is endocytosed and that apical endocytosis is essential for cell shape change and for bending of the neural plate, but how this process is regulated remains entirely unknown. In Aim I of this application, we propose experiments that will determine the role of Shroom3 in triggering apical endocytosis and will elucidate the protein machinery driving endocytosis in Shroom3 expressing cells. The central nervous system of vertebrates develops initially as a flat sheet of cells that will roll up and seal shut to form the hollow neural tube. Several potentially force-generating mechanisms have been identified that contribute to neural tube closure, including apical constriction, convergent extension, and a poorly-defined pushing force generated by the neighboring epidermal cells. Despite this progress, we still have no comprehensive understanding of how these different force-generating "engines" function cooperatively to effect neural tube closure. In Aim II, we will quantify the behavior of both neural and non-neural epithelial cells during neural tube closure in large numbers of wild-type embryos and in embryos lacking Shroom3 function. Shroom3 is essential for epithelial cell shape change during development of the neural tube, the lens and the gut. Because Shroom3 is sufficient to induce dramatic cell shape changes, understanding the transcriptional control of this gene will be essential to any comprehensive understanding of epithelial sheet- bending in vertebrate embryos. Experiments in Aim III will identify transcriptional activators of Shroom3 expression and lay the foundation for a gene regulatory network governing epithelial morphogenesis in vertebrates. Page 5 PUBLIC HEALTH RELEVANCE: The long-term goal of our research is to understand the molecular and cellular mechanisms governing neural tube closure in vertebrate embryos. The human central nervous system develops initially as a flat sheet of cells in the early embryo and subsequently rolls up and seals shut to form a tube. Failure of this rolling and sealing process leads to spina bifida and other birth defects called Neural Tube Defects. The application proposes experiments centered in three areas. 1) We will examine the basic cell biological processes underlying cell shape change in the developing nervous system. 2) We will examine how changes in the shape of an individual cells impacts shape in neighboring cells and how this impacts the rolling of the tissue generally. 3) We will identify the genetic regulators of cell shape change. Together, these experiments will provide new insights into the etiology of neural tube birth defects.